CA3004741A1 - Novel methods of treating hearing loss - Google Patents
Novel methods of treating hearing loss Download PDFInfo
- Publication number
- CA3004741A1 CA3004741A1 CA3004741A CA3004741A CA3004741A1 CA 3004741 A1 CA3004741 A1 CA 3004741A1 CA 3004741 A CA3004741 A CA 3004741A CA 3004741 A CA3004741 A CA 3004741A CA 3004741 A1 CA3004741 A1 CA 3004741A1
- Authority
- CA
- Canada
- Prior art keywords
- hearing loss
- treatment
- administration
- animal
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000016354 hearing loss disease Diseases 0.000 title claims abstract description 75
- 206010011878 Deafness Diseases 0.000 title claims abstract description 67
- 231100000888 hearing loss Toxicity 0.000 title claims abstract description 66
- 230000010370 hearing loss Effects 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 58
- 238000011282 treatment Methods 0.000 claims abstract description 71
- 230000000694 effects Effects 0.000 claims abstract description 36
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 30
- 230000003185 calcium uptake Effects 0.000 claims abstract description 15
- 230000030833 cell death Effects 0.000 claims abstract description 12
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 6
- 101000801296 Homo sapiens Protein O-mannosyl-transferase TMTC4 Proteins 0.000 claims description 66
- 102100033737 Protein O-mannosyl-transferase TMTC4 Human genes 0.000 claims description 65
- 210000004027 cell Anatomy 0.000 claims description 58
- 241001465754 Metazoa Species 0.000 claims description 45
- 210000002768 hair cell Anatomy 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 17
- 102100030013 Endoribonuclease Human genes 0.000 claims description 10
- 230000004044 response Effects 0.000 claims description 9
- -1 nitroxides Chemical class 0.000 claims description 6
- 231100000199 ototoxic Toxicity 0.000 claims description 6
- 230000002970 ototoxic effect Effects 0.000 claims description 6
- 101000975407 Caenorhabditis elegans Inositol 1,4,5-trisphosphate receptor itr-1 Proteins 0.000 claims description 5
- 101000975393 Drosophila melanogaster Inositol 1,4,5-trisphosphate receptor Proteins 0.000 claims description 5
- 230000001640 apoptogenic effect Effects 0.000 claims description 5
- BHTRKEVKTKCXOH-UHFFFAOYSA-N Taurochenodesoxycholsaeure Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCCS(O)(=O)=O)C)C1(C)CC2 BHTRKEVKTKCXOH-UHFFFAOYSA-N 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 4
- 108091052345 ryanodine receptor (TC 1.A.3.1) family Proteins 0.000 claims description 4
- 206010011903 Deafness traumatic Diseases 0.000 claims description 3
- 208000002946 Noise-Induced Hearing Loss Diseases 0.000 claims description 3
- 230000004094 calcium homeostasis Effects 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 239000004031 partial agonist Substances 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 claims description 2
- MPYLDWFDPHRTEG-PAAYLBSLSA-N (3e,5s,8r,9s,10r,13s,14s)-3-(2-aminoethoxyimino)-10,13-dimethyl-1,2,4,5,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthrene-6,17-dione Chemical compound C1\C(=N\OCCN)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC(=O)[C@H]21 MPYLDWFDPHRTEG-PAAYLBSLSA-N 0.000 claims description 2
- NOHQEAFAESMMDX-UHFFFAOYSA-N 1-[4-(8-amino-3-tert-butylimidazo[1,5-a]pyrazin-1-yl)naphthalen-1-yl]-3-[3-(trifluoromethyl)phenyl]urea Chemical compound C=12C(N)=NC=CN2C(C(C)(C)C)=NC=1C(C1=CC=CC=C11)=CC=C1NC(=O)NC1=CC=CC(C(F)(F)F)=C1 NOHQEAFAESMMDX-UHFFFAOYSA-N 0.000 claims description 2
- BLZVCIGGICSWIG-UHFFFAOYSA-N 2-aminoethoxydiphenylborane Chemical compound C=1C=CC=CC=1B(OCCN)C1=CC=CC=C1 BLZVCIGGICSWIG-UHFFFAOYSA-N 0.000 claims description 2
- IVQVBMWPWPTSNO-UHFFFAOYSA-N 2-hydroxy-6-[5-(4-methylpiperazine-1-carbonyl)thiophen-2-yl]naphthalene-1-carbaldehyde Chemical group C1CN(C)CCN1C(=O)C1=CC=C(C=2C=C3C=CC(O)=C(C=O)C3=CC=2)S1 IVQVBMWPWPTSNO-UHFFFAOYSA-N 0.000 claims description 2
- WJNBSTLIALIIEW-UHFFFAOYSA-N 2-n-(3h-benzimidazol-5-yl)-4-n-(5-cyclopropyl-1h-pyrazol-3-yl)pyrimidine-2,4-diamine Chemical compound C1CC1C1=NNC(NC=2N=C(NC=3C=C4N=CNC4=CC=3)N=CC=2)=C1 WJNBSTLIALIIEW-UHFFFAOYSA-N 0.000 claims description 2
- PJZLSMMERMMQBJ-UHFFFAOYSA-N 3,5-ditert-butylbenzene-1,2-diol Chemical compound CC(C)(C)C1=CC(O)=C(O)C(C(C)(C)C)=C1 PJZLSMMERMMQBJ-UHFFFAOYSA-N 0.000 claims description 2
- DVKVZPIRWWREJC-UHFFFAOYSA-N 4-chloro-3-ethylphenol Chemical compound CCC1=CC(O)=CC=C1Cl DVKVZPIRWWREJC-UHFFFAOYSA-N 0.000 claims description 2
- UZFMOKQJFYMBGY-UHFFFAOYSA-N 4-hydroxy-TEMPO Chemical group CC1(C)CC(O)CC(C)(C)N1[O] UZFMOKQJFYMBGY-UHFFFAOYSA-N 0.000 claims description 2
- 229930184976 Adenophostin Natural products 0.000 claims description 2
- RENVITLQVBEFDT-MZQFDOALSA-N Adenophostin A Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]1OP(O)(O)=O)N1C=2N=CN=C(C=2N=C1)N)[C@H]1O[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H]1O RENVITLQVBEFDT-MZQFDOALSA-N 0.000 claims description 2
- GVGVYDCVFBGALZ-UHFFFAOYSA-N CDN1163 Chemical compound C1=CC(OC(C)C)=CC=C1C(=O)NC1=CC=CC2=CC=C(C)N=C12 GVGVYDCVFBGALZ-UHFFFAOYSA-N 0.000 claims description 2
- PRWSIEBRGXYXAJ-UHFFFAOYSA-N GSK2656157 Chemical group CC1=CC=CC(CC(=O)N2C3=C(C(=C(C=4C5=C(N)N=CN=C5N(C)C=4)C=C3)F)CC2)=N1 PRWSIEBRGXYXAJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000002147 L01XE04 - Sunitinib Substances 0.000 claims description 2
- 102000017955 Regucalcin Human genes 0.000 claims description 2
- 108050007056 Regucalcin Proteins 0.000 claims description 2
- 239000000556 agonist Substances 0.000 claims description 2
- TVIVJHZHPKNDAQ-MHWRWJLKSA-N chembl3192687 Chemical compound OC1=CC=C2C=CC=CC2=C1\C=N\S(=O)(=O)C1=CC=CS1 TVIVJHZHPKNDAQ-MHWRWJLKSA-N 0.000 claims description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 2
- 238000001415 gene therapy Methods 0.000 claims description 2
- 208000014674 injury Diseases 0.000 claims description 2
- 229950005528 istaroxime Drugs 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- WINHZLLDWRZWRT-ATVHPVEESA-N sunitinib Chemical compound CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C WINHZLLDWRZWRT-ATVHPVEESA-N 0.000 claims description 2
- 229960001796 sunitinib Drugs 0.000 claims description 2
- BHTRKEVKTKCXOH-AYSJQVDDSA-N taurochenodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)C1C2C2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)CC1 BHTRKEVKTKCXOH-AYSJQVDDSA-N 0.000 claims description 2
- BHTRKEVKTKCXOH-LBSADWJPSA-N tauroursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)CC1 BHTRKEVKTKCXOH-LBSADWJPSA-N 0.000 claims description 2
- 230000008733 trauma Effects 0.000 claims description 2
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 claims description 2
- 229960001661 ursodiol Drugs 0.000 claims description 2
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 claims description 2
- 101001010783 Homo sapiens Endoribonuclease Proteins 0.000 claims 2
- 208000026091 Congenital hearing disease Diseases 0.000 claims 1
- MMWCIQZXVOZEGG-XJTPDSDZSA-N D-myo-Inositol 1,4,5-trisphosphate Chemical class O[C@@H]1[C@H](O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H](O)[C@@H]1OP(O)(O)=O MMWCIQZXVOZEGG-XJTPDSDZSA-N 0.000 claims 1
- 230000003044 adaptive effect Effects 0.000 claims 1
- 102000042094 ryanodine receptor (TC 1.A.3.1) family Human genes 0.000 claims 1
- 230000001594 aberrant effect Effects 0.000 abstract description 8
- 230000005735 apoptotic response Effects 0.000 abstract description 4
- 230000002159 abnormal effect Effects 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 description 39
- 230000004906 unfolded protein response Effects 0.000 description 30
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 19
- 229910001424 calcium ion Inorganic materials 0.000 description 19
- 210000002950 fibroblast Anatomy 0.000 description 16
- 230000013707 sensory perception of sound Effects 0.000 description 15
- 108091008038 CHOP Proteins 0.000 description 13
- 102100029145 DNA damage-inducible transcript 3 protein Human genes 0.000 description 13
- 239000003814 drug Substances 0.000 description 13
- 229940124597 therapeutic agent Drugs 0.000 description 9
- 108091006081 Inositol-requiring enzyme-1 Proteins 0.000 description 8
- HATRDXDCPOXQJX-UHFFFAOYSA-N Thapsigargin Natural products CCCCCCCC(=O)OC1C(OC(O)C(=C/C)C)C(=C2C3OC(=O)C(C)(O)C3(O)C(CC(C)(OC(=O)C)C12)OC(=O)CCC)C HATRDXDCPOXQJX-UHFFFAOYSA-N 0.000 description 8
- 230000001771 impaired effect Effects 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- IXFPJGBNCFXKPI-FSIHEZPISA-N thapsigargin Chemical compound CCCC(=O)O[C@H]1C[C@](C)(OC(C)=O)[C@H]2[C@H](OC(=O)CCCCCCC)[C@@H](OC(=O)C(\C)=C/C)C(C)=C2[C@@H]2OC(=O)[C@@](C)(O)[C@]21O IXFPJGBNCFXKPI-FSIHEZPISA-N 0.000 description 8
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 7
- 108090000397 Caspase 3 Proteins 0.000 description 7
- 229910052791 calcium Inorganic materials 0.000 description 7
- 239000011575 calcium Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000035882 stress Effects 0.000 description 7
- 102000003952 Caspase 3 Human genes 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000004907 flux Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 5
- 239000012620 biological material Substances 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 230000034994 death Effects 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 230000003938 response to stress Effects 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 229940123090 PERK inhibitor Drugs 0.000 description 4
- 230000005775 apoptotic pathway Effects 0.000 description 4
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 4
- 210000003477 cochlea Anatomy 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 125000001188 haloalkyl group Chemical group 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 238000011813 knockout mouse model Methods 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- HJGMCDHQPXTGAV-UHFFFAOYSA-N 2-(4-chlorophenoxy)-n-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide Chemical compound C1=CC(Cl)=CC=C1OCC(=O)NC1CCC(NC(=O)COC=2C=CC(Cl)=CC=2)CC1 HJGMCDHQPXTGAV-UHFFFAOYSA-N 0.000 description 3
- 101710205660 Calcium-transporting ATPase Proteins 0.000 description 3
- 101710134161 Calcium-transporting ATPase sarcoplasmic/endoplasmic reticulum type Proteins 0.000 description 3
- 101000936911 Chionoecetes opilio Sarcoplasmic/endoplasmic reticulum calcium ATPase Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 102000019027 Ryanodine Receptor Calcium Release Channel Human genes 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000008649 adaptation response Effects 0.000 description 3
- 125000003545 alkoxy group Chemical group 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 210000000133 brain stem Anatomy 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 230000008045 co-localization Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000006735 deficit Effects 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 238000003197 gene knockdown Methods 0.000 description 3
- 125000001475 halogen functional group Chemical group 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000016914 response to endoplasmic reticulum stress Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000009211 stress pathway Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 201000004384 Alopecia Diseases 0.000 description 2
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 2
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 2
- 102100023580 Cyclic AMP-dependent transcription factor ATF-4 Human genes 0.000 description 2
- 108700041152 Endoplasmic Reticulum Chaperone BiP Proteins 0.000 description 2
- 101000905743 Homo sapiens Cyclic AMP-dependent transcription factor ATF-4 Proteins 0.000 description 2
- 101100425727 Homo sapiens TMTC4 gene Proteins 0.000 description 2
- 108091008585 IP3 receptors Proteins 0.000 description 2
- 102000007640 Inositol 1,4,5-Trisphosphate Receptors Human genes 0.000 description 2
- 208000005141 Otitis Diseases 0.000 description 2
- 101150064454 TMTC4 gene Proteins 0.000 description 2
- 108010035430 X-Box Binding Protein 1 Proteins 0.000 description 2
- 102100038151 X-box-binding protein 1 Human genes 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 208000019258 ear infection Diseases 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000003676 hair loss Effects 0.000 description 2
- 230000003284 homeostatic effect Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 210000000067 inner hair cell Anatomy 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000000366 juvenile effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 210000004927 skin cell Anatomy 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N thiocyanic acid Chemical compound SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 2
- 210000003454 tympanic membrane Anatomy 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- PDURUKZNVHEHGO-UHFFFAOYSA-N 2-[6-[bis(carboxymethyl)amino]-5-(carboxymethoxy)-1-benzofuran-2-yl]-1,3-oxazole-5-carboxylic acid Chemical compound O1C=2C=C(N(CC(O)=O)CC(O)=O)C(OCC(=O)O)=CC=2C=C1C1=NC=C(C(O)=O)O1 PDURUKZNVHEHGO-UHFFFAOYSA-N 0.000 description 1
- OBKXEAXTFZPCHS-UHFFFAOYSA-N 4-phenylbutyric acid Chemical group OC(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108091005753 BiP proteins Proteins 0.000 description 1
- 208000000477 Bilateral Hearing Loss Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102000003922 Calcium Channels Human genes 0.000 description 1
- 108090000312 Calcium Channels Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 108010069176 Connexin 30 Proteins 0.000 description 1
- 208000016952 Ear injury Diseases 0.000 description 1
- 102100039328 Endoplasmin Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102100037156 Gap junction beta-2 protein Human genes 0.000 description 1
- 102100039401 Gap junction beta-6 protein Human genes 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 208000000258 High-Frequency Hearing Loss Diseases 0.000 description 1
- 101000954092 Homo sapiens Gap junction beta-2 protein Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 102000026889 Myosin VIIa Human genes 0.000 description 1
- 108010009047 Myosin VIIa Proteins 0.000 description 1
- 240000001307 Myosotis scorpioides Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000002141 Plasma Membrane Calcium-Transporting ATPases Human genes 0.000 description 1
- 108010040945 Plasma Membrane Calcium-Transporting ATPases Proteins 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101710129655 Sarcoplasmic/endoplasmic reticulum calcium ATPase Proteins 0.000 description 1
- 208000009966 Sensorineural Hearing Loss Diseases 0.000 description 1
- 208000027077 Stickler syndrome Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 208000026724 Waardenburg syndrome Diseases 0.000 description 1
- 231100000569 acute exposure Toxicity 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 210000000877 corpus callosum Anatomy 0.000 description 1
- 231100000895 deafness Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 210000000613 ear canal Anatomy 0.000 description 1
- 239000003221 ear drop Substances 0.000 description 1
- 229940047652 ear drops Drugs 0.000 description 1
- 238000013028 emission testing Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000009395 genetic defect Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 108010017007 glucose-regulated proteins Proteins 0.000 description 1
- SIXVRXARNAVBTC-UHFFFAOYSA-N gsk2606414 Chemical compound C12=C(N)N=CN=C2N(C)C=C1C(C=C1CC2)=CC=C1N2C(=O)CC1=CC=CC(C(F)(F)F)=C1 SIXVRXARNAVBTC-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000012074 hearing test Methods 0.000 description 1
- 231100000885 high-frequency hearing loss Toxicity 0.000 description 1
- 230000000971 hippocampal effect Effects 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000008216 juvenile development Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 201000006790 nonsyndromic deafness Diseases 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001992 poloxamer 407 Polymers 0.000 description 1
- 229940044476 poloxamer 407 Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000000752 vestibulocochlear nerve Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
- A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
- A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/69—Boron compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Pain & Pain Management (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Provided are methods of treating various forms of hearing loss caused by aberrant calcium fluxes in auditory cells. The methods encompass the administration of agents which prevent abnormal calcium fluxes, stabilize SERCA2b activity, or prevent ER UPR apoptotic responses. The methods include therapeutic treatments of subjects suffering from hearing loss as well as preventative treatments which protect auditory cells from cell death, for example as caused by exposure to loud noise.
Description
NOVEL METHODS OF TREATING HEARING LOSS
CROSS-REFERENCE TO RELATED APPLICATIONS: This application claims the benefit of priority to United States Provisional Application Serial Number 62/246,425 entitled "Novel Methods of Treating Hearing Loss," filed October 26, 2015, the contents which are hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT: This invention was made with government support under grant no.
N5058721 awarded by the National Institutes of Health. The government has certain rights in the invention.
Background of the Invention Hearing loss is a significant public health concern. The overall prevalence is 10.5% of the male population and 6.8% of females. Therefore, about thirty million Americans have bilateral hearing loss, based on standard hearing examinations. 18% of adults 45-64 years old, 30% of adults 65-74 years old and 47% of adults 75 years or older have hearing loss.
A full 27% of hearing loss occurs in an acute setting (ear infection, ear injury and loud brief noise), while another 23% occur from more chronic exposure to sound. The NICHD estimates that 26 million Americans between the ages of 20 and 69 have high frequency hearing loss due to exposure at work or during leisure activities. Thus, there is with this group alone a very significant population in the US who could benefit immediately from treatments that can prevent these exposure-based hearing loss cases. There are also genetic contributions to hearing loss, with 3 out of 1,000 births associated with detectable hearing impairment. The costs of hearing loss to SUBSTITUTE SHEET (RULE 26) society are also significant, for example, the average lifetime expenses for a person with prelingual significant hearing impairment average about one million dollars.
Summary of the Invention Advantageously, the inventor of the present disclosure has discovered that the death of hair cells in the ear can be caused by over-activation of the unfolded protein response.
Specifically, the action of dysregulated calcium fluxes has been implicated in the death of hair cells, which dysregulated calcium fluxes result in over-activation of apoptotic UPR processes.
Accordingly, in one aspect, the scope of the invention encompasses treatments which inhibit or reverse abnormal calcium fluxes that trigger maladaptive UPR pathways in auditory cells, including hair cells and supporting cells of the cochlea. In another aspect, the invention is directed to treatments that inhibit maladaptive UPR pathways in order to rescue hair cells from cell death. The scope of the invention encompasses therapeutic treatments for subjects suffering from various forms of hearing loss, and further encompasses preventative treatments for subjects at risk of hearing damage.
Description of the Drawings Fig. 1. Auditory brainstem response click threshold in mice at different developmental stages.
Dotted line = wild type mice, Solid line = TMTC4 homozygous knockout mice, and Dashed line = heterozygous TMTC4 +/- mice.
Fig. 2. SYBR Green measurement of ciPCR product measuring RNA expression of spliced X-box binding protein 1 (S-XBP1), the molecular chaperone BiP, the pro-apoptotic protein CHOP,
CROSS-REFERENCE TO RELATED APPLICATIONS: This application claims the benefit of priority to United States Provisional Application Serial Number 62/246,425 entitled "Novel Methods of Treating Hearing Loss," filed October 26, 2015, the contents which are hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT: This invention was made with government support under grant no.
N5058721 awarded by the National Institutes of Health. The government has certain rights in the invention.
Background of the Invention Hearing loss is a significant public health concern. The overall prevalence is 10.5% of the male population and 6.8% of females. Therefore, about thirty million Americans have bilateral hearing loss, based on standard hearing examinations. 18% of adults 45-64 years old, 30% of adults 65-74 years old and 47% of adults 75 years or older have hearing loss.
A full 27% of hearing loss occurs in an acute setting (ear infection, ear injury and loud brief noise), while another 23% occur from more chronic exposure to sound. The NICHD estimates that 26 million Americans between the ages of 20 and 69 have high frequency hearing loss due to exposure at work or during leisure activities. Thus, there is with this group alone a very significant population in the US who could benefit immediately from treatments that can prevent these exposure-based hearing loss cases. There are also genetic contributions to hearing loss, with 3 out of 1,000 births associated with detectable hearing impairment. The costs of hearing loss to SUBSTITUTE SHEET (RULE 26) society are also significant, for example, the average lifetime expenses for a person with prelingual significant hearing impairment average about one million dollars.
Summary of the Invention Advantageously, the inventor of the present disclosure has discovered that the death of hair cells in the ear can be caused by over-activation of the unfolded protein response.
Specifically, the action of dysregulated calcium fluxes has been implicated in the death of hair cells, which dysregulated calcium fluxes result in over-activation of apoptotic UPR processes.
Accordingly, in one aspect, the scope of the invention encompasses treatments which inhibit or reverse abnormal calcium fluxes that trigger maladaptive UPR pathways in auditory cells, including hair cells and supporting cells of the cochlea. In another aspect, the invention is directed to treatments that inhibit maladaptive UPR pathways in order to rescue hair cells from cell death. The scope of the invention encompasses therapeutic treatments for subjects suffering from various forms of hearing loss, and further encompasses preventative treatments for subjects at risk of hearing damage.
Description of the Drawings Fig. 1. Auditory brainstem response click threshold in mice at different developmental stages.
Dotted line = wild type mice, Solid line = TMTC4 homozygous knockout mice, and Dashed line = heterozygous TMTC4 +/- mice.
Fig. 2. SYBR Green measurement of ciPCR product measuring RNA expression of spliced X-box binding protein 1 (S-XBP1), the molecular chaperone BiP, the pro-apoptotic protein CHOP,
2 SUBSTITUTE SHEET (RULE 26) and Caspase 3 in cochlear explants from mice. WT = wild type and KO = TMTC4 knockout mice. +TG indicates explants treated with the SERCA2b inhibitor thapsigargin.
Fig. 3. Fig. 3 depicts cytosolic calcium ion Cal peaks per minute in cochleae of wild type and TMTC4 KO mice.
Fig. 4. Fig. 4 depicts cytosolic Ca2+ peak average peak height in arbitrary units for peaks in cochleae and fibroblasts.
Fig. 5. Fig. 5 depicts Cal' peak average decay time for peaks in cochleae and fibroblasts.
Fig. 6. Fig. 6. depicts the aggregate peak time course for Cal' peaks in cochleae. Solid line-wild type. Dotted line- TMTC4 KO.
Fig. 7. Fig. 7. depicts the aggregate peak time course for Cal' peaks in fibroblasts. Solid line-wild type. Dotted line- TMTC4 KO.
Fig. 8. Fig. 8 depicts the quantification of CHOP, Bip, S-XBP1, and caspase 3 mRNA
expression in cochleae of wild type mice exposed to sound at levels previously shown to cause hearing damage, two hours after such exposure. CTRL- untreated mice. N-E-noise exposed mice.
Fig. 9. Fig. 9 depicts ABR threshold to click, 8 kHz, and 16 kHz stimuli in mice treated with ISRIB (dotted line, N=3)) compared with vehicle (solid line, N=3). All values are means with error bars indicating the standard error of the mean.
Fig. 3. Fig. 3 depicts cytosolic calcium ion Cal peaks per minute in cochleae of wild type and TMTC4 KO mice.
Fig. 4. Fig. 4 depicts cytosolic Ca2+ peak average peak height in arbitrary units for peaks in cochleae and fibroblasts.
Fig. 5. Fig. 5 depicts Cal' peak average decay time for peaks in cochleae and fibroblasts.
Fig. 6. Fig. 6. depicts the aggregate peak time course for Cal' peaks in cochleae. Solid line-wild type. Dotted line- TMTC4 KO.
Fig. 7. Fig. 7. depicts the aggregate peak time course for Cal' peaks in fibroblasts. Solid line-wild type. Dotted line- TMTC4 KO.
Fig. 8. Fig. 8 depicts the quantification of CHOP, Bip, S-XBP1, and caspase 3 mRNA
expression in cochleae of wild type mice exposed to sound at levels previously shown to cause hearing damage, two hours after such exposure. CTRL- untreated mice. N-E-noise exposed mice.
Fig. 9. Fig. 9 depicts ABR threshold to click, 8 kHz, and 16 kHz stimuli in mice treated with ISRIB (dotted line, N=3)) compared with vehicle (solid line, N=3). All values are means with error bars indicating the standard error of the mean.
3 SUBSTITUTE SHEET (RULE 26) Detailed Description of the Invention The invention encompasses various methods of treating a hearing loss condition in an animal subject. As used herein, "treatment" of a hearing loss condition means the administration of one or more chemical or biological agents to the animal subject in order to: alleviate causative processes underlying the hearing loss; reduce one or more symptoms or measures of hearing loss; prevent, ameliorate, inhibit, or reverse the loss of hair cells; and/or otherwise inhibit processes that negatively affect auditory ability.
Some of the treatments described herein are directed to the modulation of a target enzyme, process, or the like. As used herein, "modulate" means to act by an agent to regulate, to control or to change the activity of the target, including enhancement of the target.
A "hearing loss condition," as used herein, broadly encompasses any damage to the auditory systems, organs, and cells or any impairment of an animal subject's ability to hear sound, as measured by standard methods and assessments known in the art, for example otoacoustic emission testing, pure tone testing, and auditory brainstem response testing.
Exemplary hearing loss conditions include noise-induced hearing loss, resulting from chronic or acute exposure to loud noise (e.g. noise above 75 decibels), for example occupational noise such as construction noise, aircraft noise, and military noise, or recreational noise such as experienced by musicians, concert attendees, or racetrack attendees. Hearing loss conditions further include various forms of age-related hearing loss, as known in the art. Hearing loss conditions further encompass various hearing impairments resulting from genetic or inherited factors, for example, congenital hearing loss, Stickler syndrome, Waardenburg syndrome, and non-syndromic hearing loss, including hearing pathologies associated with genes such as GJB2 and GJB6. Hearing loss
Some of the treatments described herein are directed to the modulation of a target enzyme, process, or the like. As used herein, "modulate" means to act by an agent to regulate, to control or to change the activity of the target, including enhancement of the target.
A "hearing loss condition," as used herein, broadly encompasses any damage to the auditory systems, organs, and cells or any impairment of an animal subject's ability to hear sound, as measured by standard methods and assessments known in the art, for example otoacoustic emission testing, pure tone testing, and auditory brainstem response testing.
Exemplary hearing loss conditions include noise-induced hearing loss, resulting from chronic or acute exposure to loud noise (e.g. noise above 75 decibels), for example occupational noise such as construction noise, aircraft noise, and military noise, or recreational noise such as experienced by musicians, concert attendees, or racetrack attendees. Hearing loss conditions further include various forms of age-related hearing loss, as known in the art. Hearing loss conditions further encompass various hearing impairments resulting from genetic or inherited factors, for example, congenital hearing loss, Stickler syndrome, Waardenburg syndrome, and non-syndromic hearing loss, including hearing pathologies associated with genes such as GJB2 and GJB6. Hearing loss
4 SUBSTITUTE SHEET (RULE 26) conditions further include hearing loss associated with disease conditions, such as ear infection, meningitis, autoimmune disease, and other conditions known in the art.
Additionally, hearing loss conditions can include hearing impairment caused by acute or chronic exposure to ototoxic agents, i.e. species which damage hearing, for example such as cisplatin, gentamicin, toluene, lead, and other species known in the art. Also, hearing loss conditions include hearing impairment associated with trauma, for example, resulting from head injuries.
Hearing loss conditions also include any reduction in the function or number of auditory cells, including hair cells, cochlear cells, or supporting cells thereof. Hair cells include inner hair cells and outer hair cells. Cochlear cells include neurons of the vestibulocochlear nerve and other cells associated with the perception of sound.
In various embodiments, the treatments of the invention may encompass therapeutic treatments to alleviate a hearing loss condition in an individual animal suffering therefrom.
Therapeutic treatments include those which reduce or reverse the severity of a hearing loss condition: improve the function of auditory cells, including hair cells; or which increase or preserve the number of auditory cells, including hair cells.
Further, the scope of the invention includes preventative treatments which arrest or slow progressive hearing loss conditions or which prophylactically protect subjects from experiencing a hearing loss condition. In one embodiment, the treatments of the invention are administered to an individual at risk for a hearing loss condition. For example, preventative treatments may be administered to aging subjects (e.g. subjects over 50, 55, 60, or 65 years of age), or subjects having genetic disposition for hearing loss. In some embodiments, the preventative treatments are administered to subjects that are imminently or potentially to be exposed to ototoxic agents, SUBSTITUTE SHEET (RULE 26) which are being exposed to ototoxic agents, or which have recently been exposed to ototoxic agents, for example, patients undergoing the administration of drugs having potential ototoxic side effects. Furthermore, in some embodiments, the preventative treatments of the invention are administered to subjects prior to, during, and/or shortly after exposure to excessive noise, for example workers (e.g. construction and pilots or airport workers), military personnel, and musicians or concertgoers.
The various treatments described herein encompass the administration of one or more therapeutic agents. The "therapeutic agent," as used herein may comprise a small molecule chemical species. It will be understood that the administration of specific small molecule agents described herein also includes the administration of analogs, variants, and derivatives of the disclosed compounds. The therapeutic agent may also comprise a biological species such as a polypeptide, antibody, nucleic acid, or hybrids thereof. Such administration of therapeutic agents described herein will be understood to comprise the administration of a therapeutically effective amount of the therapeutic agent, as known in the art.
It will be understood that such "administration," as used herein may encompass, oral, transdermal, intraperitoneal, subcutaneous, intravenous, or other administration routes, as appropriate for the particular therapeutic agent being administered. In one embodiment, the one or more agents is administered by infusion into the ear. Ear infusion, wherein the agent crosses the tympanic membrane and contacts auditory cells, such as hair cells, allows directed delivery to the ear and minimizes side effects that may occur with systemic administration. Such delivery of the agent may be accomplished by any transtympanic drug delivery methods known in the art.
Exemplary transtympanic drug delivery methods include iontophoretic systems, for example, as described in United States Patent Number 7,840,260, entitled, "Iontophoretic intra-tympanic SUBSTITUTE SHEET (RULE 26) drug delivery system," by Eply. Another potential transtympanic delivery system includes the use of membrane-permeating carriers, such as described in United States Patent Number 8,822,410, entitled "Tympanic membrane permeating ear drops and uses thereof,"
by Simons.
Another delivery system includes the use of hydrogels infused with therapeutic agents and chemical penetration enhances, for example, penta- block copolymer poloxamer 407¨
polybutylphosphoester gels as described in the article: "Treatment of otitis -media by transtympanic delivery of antibiotics", by Rong Yang et al., Science Translational Medicine 8:
356 (2016). Additional transtympanic drug deliver methods include transtympanic injection or by surgical implantation of drug eluting pellets of like structures by tympanomeatal flap procedure.
It will be further understood that such administration includes the use of pharmaceutically acceptable carriers or excipients, as known in the art, for example to optimize or modulate the bioavailability or pharmacokinetics of the agent, facilitate delivery to target tissues, and/or preserve the therapeutic agent.
The various methods described herein are directed to the treatment of a hearing loss condition in an animal subject in need of such treatment. The animal subject may be of any species of animal, for example a human being, e.g. a human patient. The treatments of the invention may also be directed to non-human animals such as dogs, cats, rats, mice, horses, pigs, cows, and other species, for example, animals treated in a veterinary context or experimental test animals.
Biological Mechanisms. As described in the Example below, the various embodiments of the invention are based on the discovery that the unfolded protein response, mediated by calcium SUBSTITUTE SHEET (RULE 26) fluxes, is implicated in the death of hair cells. Specifically, the inventor of the present disclosure has determined that drastic hearing loss occurs in mice wherein the gene TMTC4 has been knocked out. TMTC4 is a protein that modulates the action of calcium pump SERCA2b. When TMTC4 activity is reduced or ablated, SERCA2b activity is dysregulated, resulting in abnormal and excessive calcium fluxes from the endoplasmic reticulum. This aberrant calcium status initiates and enhances the the unfolded protein response (UPR), leading to apoptosis-mediated cell death in hair cells and associated auditory cells. Accordingly, the treatments of the invention are directed to the various processes and pathways implicated in this discovery.
SERAC2b. The Sarcoplasmic/Endoplasmic Reticulum Calcium Atpase 2b (SERAC2b) calcium pump is responsible for maintaining homeostatic calcium levels within the ER.
Impairment or loss of SERCA2b activity in auditory cells triggers ER stress via over-activation of the unfolded protein response, leading to apoptosis and auditory cell death. Accordingly, in one aspect, the invention encompasses treatments directed to the modulation of SERCA2b activity. In one embodiment, the invention comprises treatments which enhance, increase, or restore SERCA2b activity, for example, in subjects having reduced (e.g. below normal) SERCA2b activity in auditory cells. In another embodiment, the invention includes treatments which stabilize SERCA2b activity, i.e. treatments which result in normal SERCA2b activity over time, as observed in normal subjects not having a hearing loss condition. For example, in one embodiment, the treatment reduces aberrant calcium fluxes into or out of the ER compartment.
In one embodiment, the treatment results in the restoration of normal SERCA2b activity, e.g.
SERCA2b activity which is within the normal range or which is closer to normal activity, compared to that untreated subjects. In one embodiment, the treatment results in normalized SUBSTITUTE SHEET (RULE 26) calcium status within the ER. In one embodiment, the treatment results in reduced or inhibited frequency and/or magnitude of aberrant calcium fluxes from the ER.
The scope of the invention also includes modulation of other SERCA calcium pumps.
Exemplary treatments include the administration of agents which modulate SERCA2b activity. In one embodiment, the invention comprises the administration of CDN1163, a known modulator of SERCA2b activity. Additional exemplary SERCA2b-enhancing agents include nitroxides such as 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol), ursodeoxycholic acid, and tauroursodeoxycholic acid. In another embodiment, the invention comprises the administration of an agent described in PCT Patent Application Publication Number WO/2010088450, by Zsebo and Dahl, entitled "Methods of treating diseases associated with the modulation of serca," for example 1110 R3 _7E
A
wherein A and B are each, independently, H, halo, hydroxyl, al.koxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, azido, or -NO2; wherein at least one of A and B is not 1-1;
E is H, F, Br, 1, hydroxyl, alkoxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, or azido; R.3 is H or alkyl of 1-3 carbons, Q is methyl, or R.3 and Q are joined together to form. a 5- 6 membered ring; and v, R.2 and G are selected from (i) and (ii) as follows: (i) v is 0, R2 is 1-1 or alkyl of 1-3 carbons; and G is 1-1, halo, hydroxyl, alkoxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, azido, or -NO2, Or G is joined together with R2 to form a 5-6 membered ring; and (ii) v is 1 to 3, R2 is 1-1 or alkyl of 1-3 carbons; and G is 1-1, halo, hydroxyl, alkoxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, azido, or -NO2. Further exemplary SERCA modulators include, for example, istaroxime, NOS, SUBSTITUTE SHEET (RULE 26) TUDCA and regucalcin, as described in PCT Patent Application Publication Number WO/2012129066, by Fu and Hotamisligil, entitled "New targets for treatment of ER stress,"
TMTC4 activity. TMTC4, as demonstrated herein, binds to and modulates the activity of the SERCA2b calcium pump. In one embodiment, the invention comprises gene therapy treatments which augment reduced TMTC4 activity or which replace missing TMTC4 activity in individuals having TMTC4 deficit, for example in subjects, including newborns or juveniles, that have a genetic defect in TMTC4 expression or activity. In another embodiment, the invention comprises the administration of an agent comprising a TMTC4 agonist which enhances TMTC4 activity.
Calcium Homeostasis. In another aspect, the invention encompasses the modulation of proteins implicated in the general regulation of Ca2+ fluxes. Homeostatic regulation of Ca2+ fluxes has a positive role in preventing ER stress and reduces the activation of cell death mechanisms, and thus may prevent damage to auditory cells. In one aspect, the invention is directed to the administration of agents known in the art which maintain or restore calcium homeostasis in cells and/or the ER compartment or which ameliorate the aberrant calcium fluxes that auditory cell death.
In one embodiment, the invention encompasses the administration of modulators of the plasma membrane calcium ATPase ATP2b. Exemplary ATP2b modulators include vanadate and eosin, and inhibitors in the caloxin family of peptides, for example as described in Chaudhary et al., "Caloxin: a novel plasma membrane Ca2+ pump inhibitor," Am J Physiol Cell Physiol. 2001 Apr;280(4):C1027-30.
SUBSTITUTE SHEET (RULE 26) In one embodiment, the invention encompasses the administration of modulators of the inositol triphosphate receptor, or InsP3R. Exemplary InsP3R modulators include: adenophostin, 2-aminoethoxydiphenylborate; and 2-0-modified IP3 analogs that are partial agonists of IP3R, for example, the compounds described in Rossi et al., 2009, "Synthetic partial agonists reveal key steps in IP3 receptor activation," Nature Chem Bio 5:631-39.
In one embodiment, the invention encompasses the administration of modulators of the ryanodine receptor. The ryanodine receptors are intracellular calcium channels that are implicated in various processes involving the release of calcium ions from organelles.
Exemplary modulators of the ryanodine receptor include 4-chloro-3-ethylphenol, trilfuoperazine, and 3,5-Di-t-Butylcatechol.
UPR Modulators. The results presented herein demonstrate that hearing loss is linked to the death of auditory cells, including hair cells and/or supporting cells, wherein over-activation of the UPR, resulting from aberrant calcium fluxes, initiates apoptotic pathways and auditory cell death. In one aspect, the invention is directed to treatments that modulate UPR signaling molecules. Accordingly, the modulation of UPR response may be utilized to inhibit over-activation of the UPR in hair cells.
UPR responses are mediated via signaling molecules such as the inositol requiring enzyme 1 (IRE1) and the double stranded RNA-activated protein kinase-like ER
kinase (PERK).
IRE1 is a transmembrane protein of the ER which senses ER stress and initiates adaptive responses. However, prolonged IRE1 activity promotes apoptotic pathways and cell death.
SUBSTITUTE SHEET (RULE 26) Likewise, PERK resides in the ER membrane and regulates the adaptive and apoptotic responses through complex regulatory pathways.
In one aspect, the invention encompasses the treatment of a hearing loss condition by administration of agents that (1) promote UPR adaptive responses; and/or (2) inhibit UPR-mediated apoptotic pathway. In one embodiment, the invention comprises the administration of an IRE1 modulator. In one embodiment, the IRE1 modulator is an IRE1 inhibitor.
In one embodiment, the invention comprises the administration of a PERK modulator. In one embodiment, the PREK modulator is a PERK inhibitor. Exemplary IRE1 modulators include MKC-3946, STF-083010, 4t8C. APY29, 1NM-PPI, KIRA3, and KIRA6. Exemplary PERK
modulators include GSK2656157, GSK2606414, ISR1B (Integrate d Stress Response inhibitor), or trans-N,N'-(cyclohexane-1,4-diyebis(2-(4-chiorophenoxy)acetamide), and sunitinib. In one embodiment, the invention comprises the administration of a Bip enhancing agent. In one embodiment, the Bip enhancing agent is 2-(3,4-Dihydroxypheny1)-2-oxoethyl ester thiocyanic acid, also known as BiP protein Inducer X or BIX. In one embodiment, the invention comprises the administration of an ER stress response reducing agent. In one embodiment, the ER stress response reducing agent is 4-Phenylbutyric acid (4-PBA).
Screening Tools. The invention further encompasses biological materials derived from TMTC4-impaired materials, including whole animals, and cells or tissue explants derived from such animals As used herein, "TMTC4 impaired biological material" means an organism, tissue explant, or cell wherein the transcription, translation or activity of the TMTC4 gene or protein has been substantially impaired or repressed therein relative to wild type controls, including by double knockout mutations, knockdown mutations, dominant negative mutants, and RNAi SUBSTITUTE SHEET (RULE 26) induced gene knockdown. In one embodiment, the invention comprises a TMTC4 impaired animal. In one embodiment, the invention comprises cells derived from a TMTC4 impaired animal, for example, cultured cells. In one embodiment, the cultured cells are fibroblast cells derived from skin cells. In another embodiment, the invention comprises a tissue explant derived from an TMTC4 impaired animal. In one embodiment, the explant is a cochlear explant. In one embodiment, the TMTC4 impaired material is or is derived from a mouse.
In one embodiment, the invention encompasses a screening method wherein TMTC4 impaired biological materials are used to identify novel therapeutic agents or to evaluate the efficacy of treatments which ameliorate a hearing loss condition, such as the death of hair cells, hearing loss symptoms, and other phenotypes associated with TMTC4 knockdown.
For example, as depicted in Example 1, early hearing is intact in TMTC4 KO mice at developmental stage P13, with nearly complete hearing observed by about developmental stage P26, a period of about two weeks. Accordingly, the TMTC4 KO animals of the invention provide an ideal model for assessing treatments on short time scales. An exemplary screening method of the invention comprises the steps of: (1) administering a putative hearing loss treatment to TMTC4 impaired biological materials, comprising animals or materials derived therefrom; (2) assessing a measure of a hearing loss condition, for example, hearing ability, a symptom of hearing loss, or a process implicated in hearing loss (e.g. aberrant calcium flux, apoptotic UPR markers, hair cell death etc.); and (3) comparing the measure of the hearing loss condition observed in the treated biological materials against a control (e.g. similar untreated materials) to assess whether the putative hearing loss condition treatment was effective in preventing, ameliorating, or otherwise reducing the incidence or severity of the hearing loss condition in the treated materials. For example, in one embodiment, the animal is treated or materials are derived from the animal SUBSTITUTE SHEET (RULE 26) during the juvenile phase wherein hearing impairment develops in such animals.
In one embodiment, the animal is a TMTC4 KO mouse and the treatment is administered during the juvenile development period of about P13 to about P26.
EXAMPLE 1. TMTC4 Knockout Mice and the role of calcium regulation and the UPR
in hair cell death.
TMTC4 knockout materials. A TMTC4 heterozygous knockout (KO) mouse was generated by inserting a LacZ and Neomycin fusion construction in place of exons 1 through 3 of the TMTC4 gene. Heterogeneous TMTC4 +/- mice were generated therefrom.
Additionally, fibroblast cell cultures were generated from skin cells of the TMTC4 KO and heterogeneous TMTC4 +/- mice.
Anatomical Analysis. Analysis of the brains of the TMTC4 KO animals demonstrated that the corpus callosum, along with the anterior commissure and hippocampal commissure, the three main cerebral commissures in the brain were intact. Disrupted cochlear architecture was observed in the TMTC4 KO mice.
Hearing Ability. Hearing ability (as measured by standard hearing threshold testing) was measured at various developmental stages in the homozygous TMTC4 KO mice, heterogeneous TMTC4 +/- mice, and wild type mice. As depicted in Fig. 1, mice of all three genotypes had equivalent hearing ability at developmental stage P13. As mice developed, hearing ability remained stable in wild type and heterozygous TMTC4 +/- mice, but progressively deteriorated in TMTC4 KO mice, to the point of total deafness by about stage P23.
Characterization of hearing ability of P26 mice using pure tone testing, optoacoustic emission, and auditory brainstem response showed total loss of hearing in TMTC4 KO mice, even at 90 SUBSTITUTE SHEET (RULE 26) dB. This hearing loss was not confined to a particular frequency, being present with clicks and with tones varying from 8 to 32 kHz.
Hearing loss in the TMTC4 KO mice appears to be caused by cell death in the cochlea. Testing cochlear explants from KO and WT mice demonstrates broad-based loss of outer hair and inner hair cells. Hair cell abundance at the cochlear base was measured in mice of varying developmental stages by staining for myosin VIIa. TMTC4 KO mice demonstrated progressive hair cell loss, beginning at about developmental stage P10, with total loss of hair cells observed by developmental stage P45.
TMTC4 is localized to the endoplasmic reticulum. C-myc antibodies used to tag showed co-localization with the ER marker GRP94 in HEK cells. This co-localization was also shown in the brains of juvenile mice. This co-localization implicates over-activation of the ER
stress response in cell death in the cochlea. Co-immunoprecipitation in HEK
cells also showed TMTC4 to be in association with SERCA2b.
ER Stress is over-activated in TMTC4 KO cells. Fibroblast cells were generated from TMTC4 KO mice and wild type mice. One group of cells was treated with the SERCA2b inhibitor thapsigargin. The expression of UPR stress pathway genes spliced X-box binding protein 1 (S-XBP1), the molecular chaperone BiP, and the pro-apoptotic protein CHOP were measured by mRNA quantification. The expression of the UPR stress response markers was low and was about equal in untreated wild type and TMTC4 KO cells. However, in the thapsigargin treated cells, expression of the UPR stress response markers was substantially elevated in the TMTC4 KO cells compared to wild type cells.
SUBSTITUTE SHEET (RULE 26) In fibroblasts, the abundance of CHOP-expressing wild type and TMTC4 KO cells was quantified by CHOP antibody staining and flow cytometry. Increased activation of CHOP was observed in the TMTC4 KO cells, with 13% of wild type cells showing CHOP
expression and 51% of TMTC4 KO cells having CHOP expression.
In fibroblasts treated with thapsigargin, the treated TMTC4 KO cells had substantially higher levels of CHOP and cleaved caspase 3 protein than treated wild type cells.
In another experiment, cochlear explants from TMTC4 KO and wild type mice were extracted. One group of explants was treated with the SERCA2b inhibitor thapsigargin. The expression of UPR stress pathway genes S-XBP1 BiP, CHOP, and the apoptotic gene Caspase 3 were measured by mRNA quantification (Fig. 2). In the untreated explants, the expression of the UPR stress response markers and of Caspase3 were about equal. However, in the thapsigargin treated explants, expression of the UPR stress response markers and of Caspase 3 was substantially elevated.
These results indicate over-activation of the ER stress pathway, and resulting apoptosis, in TMTC4 KO cells and in the cochlea of TMTC4 KO mice.
Calcium Fluxes and TMTC4. In another experiment, the dynamics of calcium ion flux were measured in cells and cochlear explants. Cytosolic calcium ion concentration was measured in both fibroblasts and neonatal cochleae of wild type and TMTC4 knockout mice by ratiometric dye fluorescence during spontaneous (cochleae) and ATP-stimulated (fibroblasts) calcium ion peak activity. Peak frequency was greater in KO cochleae (Fig. 3).
Peak height was SUBSTITUTE SHEET (RULE 26) slightly higher in KO cochleae, and indistinguishable in fibroblasts (Fig. 4).
Decay time was significantly longer in both KO cochleae and fibroblasts (Fig 5). Aggregate peak time course analysis of calcium ion peaks showed identical initial kinetics and subsequent significantly delayed return to baseline in the KO cochleae (Fig. 6) and fibroblasts (Fig.
7).
These experiments demonstrated that absence of TMTC4 leads to increased fluxes of calcium ions into the cytoplasm (and decreased concentration in the ER) and reduced ability to clear calcium ions from the cytoplasm. This would activate ER stress and resulting apoptosis. These findings demonstrate the relationship between calcium ion flux, ER stress and eventual hearing loss.
Calcium Gradients. In one experiment, quantification of Ca2+ was performed in intracellular compartments of fibroblasts by Mag-FURA-2 imaging. In wild type fibroblasts, an area of high Ca2+ concentration in the perinuclear region corresponding to the ER was observed. When treated for ten minutes with 1 micromolar ATP and 1 micromolar thapsigargin, the Ca2+ concentration of the same perinuclear region was reduced to that observed in the surrounding cytosol. Quantification of the Ca2+ gradient between ER and cytosol compartments in fibroblasts was also measured in both wild type and mice. The gradient was significantly reduced in the TMTC4 KO cells, being about 33% of that observed in wild type cells. This demonstrates that there is a significantly reduced Ca2+ gradient in KO cells consistent with baseline depletion of ER Ca2+ due to aberrant SERCA activity.
SUBSTITUTE SHEET (RULE 26) Noise Induction of UPR Apoptotic Pathways. In another experiment, exposure of wild type mice to 120 db white noise across the 8-16 kH octave band for 120 minutes resulted in substantial loss of hearing across sound frequencies at day one following exposure, with some recovery in the days thereafter. Wild type mice were subjected to the same treatment and cochleae were subsequently removed and apoptotic ER response gene expression was assessed. Noise exposure caused a significant upregulation in CHOP, BiP, spliced X-box1, and Caspase 3 expression in cochleae (Fig. 8), demonstrating the role of UPR apoptotic response in the mechanism of hearing loss.
PERK Inhibitor Reduces UPR Induction Response. In another experiment, HEK293T
cells were pretreated with a control formulation or ISRIB, a PERK inhibitor, then exposed overnight to 1 micromolar thapsigargin for 2 hours. CHOP and ATF4 mRNA levels were then assessed by qPCR. A significant and substantial reduction in ATF4 and CHOP
expression was observed in the cells treated with PERK inhibitor. This demonstrates the ability of PERK inhibitors to reduce UPR induction.
UPR Inhibition Preventatively Reduces Noise-Induced Hearing Loss. ISRIB was delivered to wild type mouse ear canals via intraperitoneal injection 4 hours before exposure to damaging noise, at 2.5 mg/kg. Subsequent hearing tests on treated mice showed that the ABR threshold to clicks at 8 kHz and 16 kHz was lower in treated mice than in untreated mice (Fig. 9). These results demonstrate that therapeutic intervention to reduce UPR apoptotic responses can preventatively protect animals from hearing loss associated with noise exposure.
All patents, patent applications, and publications cited in this specification are herein SUBSTITUTE SHEET (RULE 26) incorporated by reference to the same extent as if each independent patent application, or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.
SUBSTITUTE SHEET (RULE 26)
Additionally, hearing loss conditions can include hearing impairment caused by acute or chronic exposure to ototoxic agents, i.e. species which damage hearing, for example such as cisplatin, gentamicin, toluene, lead, and other species known in the art. Also, hearing loss conditions include hearing impairment associated with trauma, for example, resulting from head injuries.
Hearing loss conditions also include any reduction in the function or number of auditory cells, including hair cells, cochlear cells, or supporting cells thereof. Hair cells include inner hair cells and outer hair cells. Cochlear cells include neurons of the vestibulocochlear nerve and other cells associated with the perception of sound.
In various embodiments, the treatments of the invention may encompass therapeutic treatments to alleviate a hearing loss condition in an individual animal suffering therefrom.
Therapeutic treatments include those which reduce or reverse the severity of a hearing loss condition: improve the function of auditory cells, including hair cells; or which increase or preserve the number of auditory cells, including hair cells.
Further, the scope of the invention includes preventative treatments which arrest or slow progressive hearing loss conditions or which prophylactically protect subjects from experiencing a hearing loss condition. In one embodiment, the treatments of the invention are administered to an individual at risk for a hearing loss condition. For example, preventative treatments may be administered to aging subjects (e.g. subjects over 50, 55, 60, or 65 years of age), or subjects having genetic disposition for hearing loss. In some embodiments, the preventative treatments are administered to subjects that are imminently or potentially to be exposed to ototoxic agents, SUBSTITUTE SHEET (RULE 26) which are being exposed to ototoxic agents, or which have recently been exposed to ototoxic agents, for example, patients undergoing the administration of drugs having potential ototoxic side effects. Furthermore, in some embodiments, the preventative treatments of the invention are administered to subjects prior to, during, and/or shortly after exposure to excessive noise, for example workers (e.g. construction and pilots or airport workers), military personnel, and musicians or concertgoers.
The various treatments described herein encompass the administration of one or more therapeutic agents. The "therapeutic agent," as used herein may comprise a small molecule chemical species. It will be understood that the administration of specific small molecule agents described herein also includes the administration of analogs, variants, and derivatives of the disclosed compounds. The therapeutic agent may also comprise a biological species such as a polypeptide, antibody, nucleic acid, or hybrids thereof. Such administration of therapeutic agents described herein will be understood to comprise the administration of a therapeutically effective amount of the therapeutic agent, as known in the art.
It will be understood that such "administration," as used herein may encompass, oral, transdermal, intraperitoneal, subcutaneous, intravenous, or other administration routes, as appropriate for the particular therapeutic agent being administered. In one embodiment, the one or more agents is administered by infusion into the ear. Ear infusion, wherein the agent crosses the tympanic membrane and contacts auditory cells, such as hair cells, allows directed delivery to the ear and minimizes side effects that may occur with systemic administration. Such delivery of the agent may be accomplished by any transtympanic drug delivery methods known in the art.
Exemplary transtympanic drug delivery methods include iontophoretic systems, for example, as described in United States Patent Number 7,840,260, entitled, "Iontophoretic intra-tympanic SUBSTITUTE SHEET (RULE 26) drug delivery system," by Eply. Another potential transtympanic delivery system includes the use of membrane-permeating carriers, such as described in United States Patent Number 8,822,410, entitled "Tympanic membrane permeating ear drops and uses thereof,"
by Simons.
Another delivery system includes the use of hydrogels infused with therapeutic agents and chemical penetration enhances, for example, penta- block copolymer poloxamer 407¨
polybutylphosphoester gels as described in the article: "Treatment of otitis -media by transtympanic delivery of antibiotics", by Rong Yang et al., Science Translational Medicine 8:
356 (2016). Additional transtympanic drug deliver methods include transtympanic injection or by surgical implantation of drug eluting pellets of like structures by tympanomeatal flap procedure.
It will be further understood that such administration includes the use of pharmaceutically acceptable carriers or excipients, as known in the art, for example to optimize or modulate the bioavailability or pharmacokinetics of the agent, facilitate delivery to target tissues, and/or preserve the therapeutic agent.
The various methods described herein are directed to the treatment of a hearing loss condition in an animal subject in need of such treatment. The animal subject may be of any species of animal, for example a human being, e.g. a human patient. The treatments of the invention may also be directed to non-human animals such as dogs, cats, rats, mice, horses, pigs, cows, and other species, for example, animals treated in a veterinary context or experimental test animals.
Biological Mechanisms. As described in the Example below, the various embodiments of the invention are based on the discovery that the unfolded protein response, mediated by calcium SUBSTITUTE SHEET (RULE 26) fluxes, is implicated in the death of hair cells. Specifically, the inventor of the present disclosure has determined that drastic hearing loss occurs in mice wherein the gene TMTC4 has been knocked out. TMTC4 is a protein that modulates the action of calcium pump SERCA2b. When TMTC4 activity is reduced or ablated, SERCA2b activity is dysregulated, resulting in abnormal and excessive calcium fluxes from the endoplasmic reticulum. This aberrant calcium status initiates and enhances the the unfolded protein response (UPR), leading to apoptosis-mediated cell death in hair cells and associated auditory cells. Accordingly, the treatments of the invention are directed to the various processes and pathways implicated in this discovery.
SERAC2b. The Sarcoplasmic/Endoplasmic Reticulum Calcium Atpase 2b (SERAC2b) calcium pump is responsible for maintaining homeostatic calcium levels within the ER.
Impairment or loss of SERCA2b activity in auditory cells triggers ER stress via over-activation of the unfolded protein response, leading to apoptosis and auditory cell death. Accordingly, in one aspect, the invention encompasses treatments directed to the modulation of SERCA2b activity. In one embodiment, the invention comprises treatments which enhance, increase, or restore SERCA2b activity, for example, in subjects having reduced (e.g. below normal) SERCA2b activity in auditory cells. In another embodiment, the invention includes treatments which stabilize SERCA2b activity, i.e. treatments which result in normal SERCA2b activity over time, as observed in normal subjects not having a hearing loss condition. For example, in one embodiment, the treatment reduces aberrant calcium fluxes into or out of the ER compartment.
In one embodiment, the treatment results in the restoration of normal SERCA2b activity, e.g.
SERCA2b activity which is within the normal range or which is closer to normal activity, compared to that untreated subjects. In one embodiment, the treatment results in normalized SUBSTITUTE SHEET (RULE 26) calcium status within the ER. In one embodiment, the treatment results in reduced or inhibited frequency and/or magnitude of aberrant calcium fluxes from the ER.
The scope of the invention also includes modulation of other SERCA calcium pumps.
Exemplary treatments include the administration of agents which modulate SERCA2b activity. In one embodiment, the invention comprises the administration of CDN1163, a known modulator of SERCA2b activity. Additional exemplary SERCA2b-enhancing agents include nitroxides such as 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol), ursodeoxycholic acid, and tauroursodeoxycholic acid. In another embodiment, the invention comprises the administration of an agent described in PCT Patent Application Publication Number WO/2010088450, by Zsebo and Dahl, entitled "Methods of treating diseases associated with the modulation of serca," for example 1110 R3 _7E
A
wherein A and B are each, independently, H, halo, hydroxyl, al.koxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, azido, or -NO2; wherein at least one of A and B is not 1-1;
E is H, F, Br, 1, hydroxyl, alkoxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, or azido; R.3 is H or alkyl of 1-3 carbons, Q is methyl, or R.3 and Q are joined together to form. a 5- 6 membered ring; and v, R.2 and G are selected from (i) and (ii) as follows: (i) v is 0, R2 is 1-1 or alkyl of 1-3 carbons; and G is 1-1, halo, hydroxyl, alkoxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, azido, or -NO2, Or G is joined together with R2 to form a 5-6 membered ring; and (ii) v is 1 to 3, R2 is 1-1 or alkyl of 1-3 carbons; and G is 1-1, halo, hydroxyl, alkoxy of 1-3 carbons, haloalkyl of 1-3 carbons, -CN, azido, or -NO2. Further exemplary SERCA modulators include, for example, istaroxime, NOS, SUBSTITUTE SHEET (RULE 26) TUDCA and regucalcin, as described in PCT Patent Application Publication Number WO/2012129066, by Fu and Hotamisligil, entitled "New targets for treatment of ER stress,"
TMTC4 activity. TMTC4, as demonstrated herein, binds to and modulates the activity of the SERCA2b calcium pump. In one embodiment, the invention comprises gene therapy treatments which augment reduced TMTC4 activity or which replace missing TMTC4 activity in individuals having TMTC4 deficit, for example in subjects, including newborns or juveniles, that have a genetic defect in TMTC4 expression or activity. In another embodiment, the invention comprises the administration of an agent comprising a TMTC4 agonist which enhances TMTC4 activity.
Calcium Homeostasis. In another aspect, the invention encompasses the modulation of proteins implicated in the general regulation of Ca2+ fluxes. Homeostatic regulation of Ca2+ fluxes has a positive role in preventing ER stress and reduces the activation of cell death mechanisms, and thus may prevent damage to auditory cells. In one aspect, the invention is directed to the administration of agents known in the art which maintain or restore calcium homeostasis in cells and/or the ER compartment or which ameliorate the aberrant calcium fluxes that auditory cell death.
In one embodiment, the invention encompasses the administration of modulators of the plasma membrane calcium ATPase ATP2b. Exemplary ATP2b modulators include vanadate and eosin, and inhibitors in the caloxin family of peptides, for example as described in Chaudhary et al., "Caloxin: a novel plasma membrane Ca2+ pump inhibitor," Am J Physiol Cell Physiol. 2001 Apr;280(4):C1027-30.
SUBSTITUTE SHEET (RULE 26) In one embodiment, the invention encompasses the administration of modulators of the inositol triphosphate receptor, or InsP3R. Exemplary InsP3R modulators include: adenophostin, 2-aminoethoxydiphenylborate; and 2-0-modified IP3 analogs that are partial agonists of IP3R, for example, the compounds described in Rossi et al., 2009, "Synthetic partial agonists reveal key steps in IP3 receptor activation," Nature Chem Bio 5:631-39.
In one embodiment, the invention encompasses the administration of modulators of the ryanodine receptor. The ryanodine receptors are intracellular calcium channels that are implicated in various processes involving the release of calcium ions from organelles.
Exemplary modulators of the ryanodine receptor include 4-chloro-3-ethylphenol, trilfuoperazine, and 3,5-Di-t-Butylcatechol.
UPR Modulators. The results presented herein demonstrate that hearing loss is linked to the death of auditory cells, including hair cells and/or supporting cells, wherein over-activation of the UPR, resulting from aberrant calcium fluxes, initiates apoptotic pathways and auditory cell death. In one aspect, the invention is directed to treatments that modulate UPR signaling molecules. Accordingly, the modulation of UPR response may be utilized to inhibit over-activation of the UPR in hair cells.
UPR responses are mediated via signaling molecules such as the inositol requiring enzyme 1 (IRE1) and the double stranded RNA-activated protein kinase-like ER
kinase (PERK).
IRE1 is a transmembrane protein of the ER which senses ER stress and initiates adaptive responses. However, prolonged IRE1 activity promotes apoptotic pathways and cell death.
SUBSTITUTE SHEET (RULE 26) Likewise, PERK resides in the ER membrane and regulates the adaptive and apoptotic responses through complex regulatory pathways.
In one aspect, the invention encompasses the treatment of a hearing loss condition by administration of agents that (1) promote UPR adaptive responses; and/or (2) inhibit UPR-mediated apoptotic pathway. In one embodiment, the invention comprises the administration of an IRE1 modulator. In one embodiment, the IRE1 modulator is an IRE1 inhibitor.
In one embodiment, the invention comprises the administration of a PERK modulator. In one embodiment, the PREK modulator is a PERK inhibitor. Exemplary IRE1 modulators include MKC-3946, STF-083010, 4t8C. APY29, 1NM-PPI, KIRA3, and KIRA6. Exemplary PERK
modulators include GSK2656157, GSK2606414, ISR1B (Integrate d Stress Response inhibitor), or trans-N,N'-(cyclohexane-1,4-diyebis(2-(4-chiorophenoxy)acetamide), and sunitinib. In one embodiment, the invention comprises the administration of a Bip enhancing agent. In one embodiment, the Bip enhancing agent is 2-(3,4-Dihydroxypheny1)-2-oxoethyl ester thiocyanic acid, also known as BiP protein Inducer X or BIX. In one embodiment, the invention comprises the administration of an ER stress response reducing agent. In one embodiment, the ER stress response reducing agent is 4-Phenylbutyric acid (4-PBA).
Screening Tools. The invention further encompasses biological materials derived from TMTC4-impaired materials, including whole animals, and cells or tissue explants derived from such animals As used herein, "TMTC4 impaired biological material" means an organism, tissue explant, or cell wherein the transcription, translation or activity of the TMTC4 gene or protein has been substantially impaired or repressed therein relative to wild type controls, including by double knockout mutations, knockdown mutations, dominant negative mutants, and RNAi SUBSTITUTE SHEET (RULE 26) induced gene knockdown. In one embodiment, the invention comprises a TMTC4 impaired animal. In one embodiment, the invention comprises cells derived from a TMTC4 impaired animal, for example, cultured cells. In one embodiment, the cultured cells are fibroblast cells derived from skin cells. In another embodiment, the invention comprises a tissue explant derived from an TMTC4 impaired animal. In one embodiment, the explant is a cochlear explant. In one embodiment, the TMTC4 impaired material is or is derived from a mouse.
In one embodiment, the invention encompasses a screening method wherein TMTC4 impaired biological materials are used to identify novel therapeutic agents or to evaluate the efficacy of treatments which ameliorate a hearing loss condition, such as the death of hair cells, hearing loss symptoms, and other phenotypes associated with TMTC4 knockdown.
For example, as depicted in Example 1, early hearing is intact in TMTC4 KO mice at developmental stage P13, with nearly complete hearing observed by about developmental stage P26, a period of about two weeks. Accordingly, the TMTC4 KO animals of the invention provide an ideal model for assessing treatments on short time scales. An exemplary screening method of the invention comprises the steps of: (1) administering a putative hearing loss treatment to TMTC4 impaired biological materials, comprising animals or materials derived therefrom; (2) assessing a measure of a hearing loss condition, for example, hearing ability, a symptom of hearing loss, or a process implicated in hearing loss (e.g. aberrant calcium flux, apoptotic UPR markers, hair cell death etc.); and (3) comparing the measure of the hearing loss condition observed in the treated biological materials against a control (e.g. similar untreated materials) to assess whether the putative hearing loss condition treatment was effective in preventing, ameliorating, or otherwise reducing the incidence or severity of the hearing loss condition in the treated materials. For example, in one embodiment, the animal is treated or materials are derived from the animal SUBSTITUTE SHEET (RULE 26) during the juvenile phase wherein hearing impairment develops in such animals.
In one embodiment, the animal is a TMTC4 KO mouse and the treatment is administered during the juvenile development period of about P13 to about P26.
EXAMPLE 1. TMTC4 Knockout Mice and the role of calcium regulation and the UPR
in hair cell death.
TMTC4 knockout materials. A TMTC4 heterozygous knockout (KO) mouse was generated by inserting a LacZ and Neomycin fusion construction in place of exons 1 through 3 of the TMTC4 gene. Heterogeneous TMTC4 +/- mice were generated therefrom.
Additionally, fibroblast cell cultures were generated from skin cells of the TMTC4 KO and heterogeneous TMTC4 +/- mice.
Anatomical Analysis. Analysis of the brains of the TMTC4 KO animals demonstrated that the corpus callosum, along with the anterior commissure and hippocampal commissure, the three main cerebral commissures in the brain were intact. Disrupted cochlear architecture was observed in the TMTC4 KO mice.
Hearing Ability. Hearing ability (as measured by standard hearing threshold testing) was measured at various developmental stages in the homozygous TMTC4 KO mice, heterogeneous TMTC4 +/- mice, and wild type mice. As depicted in Fig. 1, mice of all three genotypes had equivalent hearing ability at developmental stage P13. As mice developed, hearing ability remained stable in wild type and heterozygous TMTC4 +/- mice, but progressively deteriorated in TMTC4 KO mice, to the point of total deafness by about stage P23.
Characterization of hearing ability of P26 mice using pure tone testing, optoacoustic emission, and auditory brainstem response showed total loss of hearing in TMTC4 KO mice, even at 90 SUBSTITUTE SHEET (RULE 26) dB. This hearing loss was not confined to a particular frequency, being present with clicks and with tones varying from 8 to 32 kHz.
Hearing loss in the TMTC4 KO mice appears to be caused by cell death in the cochlea. Testing cochlear explants from KO and WT mice demonstrates broad-based loss of outer hair and inner hair cells. Hair cell abundance at the cochlear base was measured in mice of varying developmental stages by staining for myosin VIIa. TMTC4 KO mice demonstrated progressive hair cell loss, beginning at about developmental stage P10, with total loss of hair cells observed by developmental stage P45.
TMTC4 is localized to the endoplasmic reticulum. C-myc antibodies used to tag showed co-localization with the ER marker GRP94 in HEK cells. This co-localization was also shown in the brains of juvenile mice. This co-localization implicates over-activation of the ER
stress response in cell death in the cochlea. Co-immunoprecipitation in HEK
cells also showed TMTC4 to be in association with SERCA2b.
ER Stress is over-activated in TMTC4 KO cells. Fibroblast cells were generated from TMTC4 KO mice and wild type mice. One group of cells was treated with the SERCA2b inhibitor thapsigargin. The expression of UPR stress pathway genes spliced X-box binding protein 1 (S-XBP1), the molecular chaperone BiP, and the pro-apoptotic protein CHOP were measured by mRNA quantification. The expression of the UPR stress response markers was low and was about equal in untreated wild type and TMTC4 KO cells. However, in the thapsigargin treated cells, expression of the UPR stress response markers was substantially elevated in the TMTC4 KO cells compared to wild type cells.
SUBSTITUTE SHEET (RULE 26) In fibroblasts, the abundance of CHOP-expressing wild type and TMTC4 KO cells was quantified by CHOP antibody staining and flow cytometry. Increased activation of CHOP was observed in the TMTC4 KO cells, with 13% of wild type cells showing CHOP
expression and 51% of TMTC4 KO cells having CHOP expression.
In fibroblasts treated with thapsigargin, the treated TMTC4 KO cells had substantially higher levels of CHOP and cleaved caspase 3 protein than treated wild type cells.
In another experiment, cochlear explants from TMTC4 KO and wild type mice were extracted. One group of explants was treated with the SERCA2b inhibitor thapsigargin. The expression of UPR stress pathway genes S-XBP1 BiP, CHOP, and the apoptotic gene Caspase 3 were measured by mRNA quantification (Fig. 2). In the untreated explants, the expression of the UPR stress response markers and of Caspase3 were about equal. However, in the thapsigargin treated explants, expression of the UPR stress response markers and of Caspase 3 was substantially elevated.
These results indicate over-activation of the ER stress pathway, and resulting apoptosis, in TMTC4 KO cells and in the cochlea of TMTC4 KO mice.
Calcium Fluxes and TMTC4. In another experiment, the dynamics of calcium ion flux were measured in cells and cochlear explants. Cytosolic calcium ion concentration was measured in both fibroblasts and neonatal cochleae of wild type and TMTC4 knockout mice by ratiometric dye fluorescence during spontaneous (cochleae) and ATP-stimulated (fibroblasts) calcium ion peak activity. Peak frequency was greater in KO cochleae (Fig. 3).
Peak height was SUBSTITUTE SHEET (RULE 26) slightly higher in KO cochleae, and indistinguishable in fibroblasts (Fig. 4).
Decay time was significantly longer in both KO cochleae and fibroblasts (Fig 5). Aggregate peak time course analysis of calcium ion peaks showed identical initial kinetics and subsequent significantly delayed return to baseline in the KO cochleae (Fig. 6) and fibroblasts (Fig.
7).
These experiments demonstrated that absence of TMTC4 leads to increased fluxes of calcium ions into the cytoplasm (and decreased concentration in the ER) and reduced ability to clear calcium ions from the cytoplasm. This would activate ER stress and resulting apoptosis. These findings demonstrate the relationship between calcium ion flux, ER stress and eventual hearing loss.
Calcium Gradients. In one experiment, quantification of Ca2+ was performed in intracellular compartments of fibroblasts by Mag-FURA-2 imaging. In wild type fibroblasts, an area of high Ca2+ concentration in the perinuclear region corresponding to the ER was observed. When treated for ten minutes with 1 micromolar ATP and 1 micromolar thapsigargin, the Ca2+ concentration of the same perinuclear region was reduced to that observed in the surrounding cytosol. Quantification of the Ca2+ gradient between ER and cytosol compartments in fibroblasts was also measured in both wild type and mice. The gradient was significantly reduced in the TMTC4 KO cells, being about 33% of that observed in wild type cells. This demonstrates that there is a significantly reduced Ca2+ gradient in KO cells consistent with baseline depletion of ER Ca2+ due to aberrant SERCA activity.
SUBSTITUTE SHEET (RULE 26) Noise Induction of UPR Apoptotic Pathways. In another experiment, exposure of wild type mice to 120 db white noise across the 8-16 kH octave band for 120 minutes resulted in substantial loss of hearing across sound frequencies at day one following exposure, with some recovery in the days thereafter. Wild type mice were subjected to the same treatment and cochleae were subsequently removed and apoptotic ER response gene expression was assessed. Noise exposure caused a significant upregulation in CHOP, BiP, spliced X-box1, and Caspase 3 expression in cochleae (Fig. 8), demonstrating the role of UPR apoptotic response in the mechanism of hearing loss.
PERK Inhibitor Reduces UPR Induction Response. In another experiment, HEK293T
cells were pretreated with a control formulation or ISRIB, a PERK inhibitor, then exposed overnight to 1 micromolar thapsigargin for 2 hours. CHOP and ATF4 mRNA levels were then assessed by qPCR. A significant and substantial reduction in ATF4 and CHOP
expression was observed in the cells treated with PERK inhibitor. This demonstrates the ability of PERK inhibitors to reduce UPR induction.
UPR Inhibition Preventatively Reduces Noise-Induced Hearing Loss. ISRIB was delivered to wild type mouse ear canals via intraperitoneal injection 4 hours before exposure to damaging noise, at 2.5 mg/kg. Subsequent hearing tests on treated mice showed that the ABR threshold to clicks at 8 kHz and 16 kHz was lower in treated mice than in untreated mice (Fig. 9). These results demonstrate that therapeutic intervention to reduce UPR apoptotic responses can preventatively protect animals from hearing loss associated with noise exposure.
All patents, patent applications, and publications cited in this specification are herein SUBSTITUTE SHEET (RULE 26) incorporated by reference to the same extent as if each independent patent application, or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.
SUBSTITUTE SHEET (RULE 26)
Claims (40)
- Claim 1. A method of treating, in an animal, a hearing loss condition, comprising the administration of a pharmaceutically effective amount of an agent which ameliorates hearing loss conditions associated with dysregulated calcium fluxes in auditory cells.
- Claim 2. The method of Claim 1, wherein the hearing loss condition is selected from the group consisting of hair cell death, age-related hearing loss, noise-induced hearing loss, genetic or inherited hearing loss, hearing loss experienced as a result of ototoxic exposure, hearing loss resulting from disease, and hearing loss resulting from trauma.
- Claim 3. The method of Claim 1, wherein the auditory cells are hair cells.
- Claim 4. The method of Claim 1, wherein the animal is a human.
- Claim 5. The method of Claim 1, wherein the treatment is a therapeutic treatment administered to an animal afflicted with a hearing loss condition.
- Claim 6. The method of Claim 1, wherein the treatment is a preventative treatment administered to an animal at risk of affliction with a hearing loss condition.
- Claim 7. The method of Claim 6, wherein the animal is likely to be, is, or has recently been exposed to excessive noise.
- Claim 8. The method of Claim 1, wherein the agent is administered systemically.
- Claim 9. The method of Claim 1, wherein the agent is administered to the auditory cells of the ear.
- Claim 10. The method of Claim 1, wherein the treatment reduces dysregulated calcium fluxes.
- Claim 11. The method of Claim 10, wherein the treatment modulates the activity of SERCA2b.
- Claim 12. The method of Claim 11, wherein the treatment enhances the activity of SERCA2b.
- Claim 13. The method of Claim 11, wherein the treatment stabilizes the activity of SERCA2b.
- Claim 14. The method of Claim 11, wherein the treatment comprises the administration of CDN1163.
- Claim 15. The method of Claim 11, wherein the treatment comprises the administration of an agent selected from the group consisting of nitroxides, 4-Hydroxy-2,2,6,6-tetrame thylpiperidine-N-oxyl, ursodeoxycholic acid, tauroursodeoxycholic acid, istaroxime, NOS, TUDCA, and regucalcin.
- Claim 16. The method of Claim 1, wherein the treatment comprises the administration of an agent which promotes calcium homeostasis in the auditory cells of the animal.
- Claim 17. The method of Claim 16, wherein the treatment comprises the administration of an agent which modulates the activity of ATP2b.
- Claim 18. The method of Claim 17, wherein the treatment comprises the administration of vanadate, eosin, or caloxin peptides.
- Claim 19. The method of Claim 18, wherein the treatment comprises the administration of an agent which modulates the activity of IP3R.
- Claim 20. The method of Claim 19, wherein the treatment comprises the administration of an agent selected from the group consisting of adenophostin, 2-aminoethoxydiphenylborate, and 2-O-modified IP3 analogs that are partial agonists of IP3R.
- Claim 21. The method of Claim 16, wherein the treatment comprises the administration of an agent which modulates the activity of the ryanodine receptor.
- Claim 22. The method of Claim 21, wherein the treatment comprises the administration of an agent selected from the group consisting of 4-chloro-3-ethylphenol, trilfuoperazine, and 3,5-Di-t-Butylcatechol.
- Claim 23. The method of Claim 22, wherein the treatment comprises the administration of an agent that ameliorates the results of dysregulated calcium fluxes in auditory cells.
- Claim 24. The method of Claim 23, wherein the treatment promotes the adaptive UPR response.
- Claim 25. The method of Claim 23, wherein the treatment reduces the apoptotic UPR response.
- Claim 26. The method of Claim 25, wherein the treatment comprises the administration of an IRE1 modulator.
- Claim 27. The method of Claim 26, wherein the IRE1 modulator is selected from the group consisting of MKC-3946, STF-083010, 41t8C, 1-NN1-PPI, APY29, KIRA3, and KIRA6.
- Claim 28. The method of Claim 25, wherein the treatment comprises the administration of a PERK modulator.
- Claim 29. The method of Claim 28, wherein the PERK modulator is selected from the group consisting of GSK2656157, GS
K2606414, and sunitinib. - Claim 30 . The method of Claim 23, wherein the treatment comprises the administration of 4-PBA or BIX.
- Claim 31. The method of Claim 1, wherein the treatment increases the activity of TMTC4 in auditory cells of the animal.
- Claim 32. The method of Claim 31, wherein the treatment comprises the administration of a TMTC4 agonist.
- Claim 33. The method of Claim 31, wherein the treatment comprises the administration of gene therapy to restore or increase TMTC4 expression in the auditory cells of the animal.
- Claim 34. An animal, wherein the expression or activity of TMTC4 is reduced in the auditory cells of the animal relative to that in wild type animals.
- Claim 35. The animal of Claim 34, wherein the animal comprises a homozygous TMTC4 knockout.
- Claim 36. A tissue explant, wherein the tissue explant is derived from the animal of Claim 34.
- Claim 37. A cell, wherein the cell is derived from the animal of Claim 34.
- Claim 38. A method of assessing the efficacy of a hearing loss treatment, comprising the steps of administering a treatment to one or more animals, tissue explants, or cells, wherein the one or more animals, tissue explants, or cells comprises an animal having reduced or absent TMTC4 expression or is derived therefrom;
measuring a measure of a hearing loss condition in the one or more animal, tissue explant, or cell;
comparing the measurement of the hearing loss condition to that in one or more control animals, tissue explants, or cells, wherein, an observed amelioration of the hearing loss condition in the treated one or more animals, tissue explants, or cells is indicative that the treatment is effective in ameliorating the hearing loss condition. - Claim 39. The method of Claim 38, wherein the animal is a mouse.
- Claim 40. The method of Claim 39, wherein the animal is a mouse at a developmental stage between P10 and P26.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562246425P | 2015-10-26 | 2015-10-26 | |
US62/246,425 | 2015-10-26 | ||
PCT/US2016/058348 WO2017074830A1 (en) | 2015-10-26 | 2016-10-22 | Novel methods of treating hearing loss |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3004741A1 true CA3004741A1 (en) | 2017-05-04 |
Family
ID=58631041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3004741A Pending CA3004741A1 (en) | 2015-10-26 | 2016-10-22 | Novel methods of treating hearing loss |
Country Status (4)
Country | Link |
---|---|
US (1) | US11957655B2 (en) |
EP (1) | EP3368037A4 (en) |
CA (1) | CA3004741A1 (en) |
WO (1) | WO2017074830A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113712945B (en) * | 2021-09-18 | 2022-08-02 | 中国人民解放军陆军军医大学第二附属医院 | Application of 4-chloro-3-ethylphenol in preparation of tumor chemotherapy drug sensitizer and anti-tumor composition |
WO2023114451A1 (en) * | 2021-12-16 | 2023-06-22 | Jacaranda Biosciences, Inc. | Upr modulators to treat hearing loss |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6806562B2 (en) * | 2013-03-15 | 2021-01-06 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Regulator of the eIF2α pathway |
-
2016
- 2016-10-22 EP EP16860555.8A patent/EP3368037A4/en active Pending
- 2016-10-22 WO PCT/US2016/058348 patent/WO2017074830A1/en active Application Filing
- 2016-10-22 CA CA3004741A patent/CA3004741A1/en active Pending
- 2016-10-22 US US15/771,195 patent/US11957655B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
US11957655B2 (en) | 2024-04-16 |
WO2017074830A1 (en) | 2017-05-04 |
EP3368037A4 (en) | 2019-06-26 |
US20180311207A1 (en) | 2018-11-01 |
EP3368037A1 (en) | 2018-09-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Brooks et al. | Reduced severity of oxygen-induced retinopathy in eNOS-deficient mice | |
Parada et al. | The microglial α7-acetylcholine nicotinic receptor is a key element in promoting neuroprotection by inducing heme oxygenase-1 via nuclear factor erythroid-2-related factor 2 | |
Yang et al. | VEGF ameliorates cognitive impairment in in vivo and in vitro ischemia via improving neuronal viability and function | |
JP2023024984A (en) | Compositions and methods for prevention and treatment of hearing loss | |
Qu et al. | Ginsenoside Rb1 prevents MPTP-induced changes in hippocampal memory via regulation of the α-synuclein/PSD-95 pathway | |
Cippitelli et al. | The novel, selective, brain-penetrant neuropeptide Y Y2 receptor antagonist, JNJ-31020028, tested in animal models of alcohol consumption, relapse, and anxiety | |
US10774328B2 (en) | Treatment for glaucoma and other eye diseases | |
Liu et al. | EETs/sEHi alleviates nociception by blocking the crosslink between endoplasmic reticulum stress and neuroinflammation in a central poststroke pain model | |
Sullivan et al. | Evoked expression of the glutamate transporter GLT-1c in retinal ganglion cells in human glaucoma and in a rat model | |
Vugler et al. | A role for the outer retina in development of the intrinsic pupillary light reflex in mice | |
Cohen et al. | Neuropeptide S in the basolateral amygdala mediates an adaptive behavioral stress response in a rat model of posttraumatic stress disorder by increasing the expression of BDNF and the neuropeptide YY1 receptor | |
Basappa et al. | The cochlea as an independent neuroendocrine organ: Expression and possible roles of a local hypothalamic–pituitary–adrenal axis-equivalent signaling system | |
Hleihil et al. | Sustained baclofen-induced activation of GABA B receptors after cerebral ischemia restores receptor expression and function and limits progressing loss of neurons | |
Holley | Application of new biological approaches to stimulate sensory repair and protection | |
US11957655B2 (en) | Methods of treating hearing loss | |
Ji et al. | Activating transcription factor 6 contributes to functional recovery after spinal cord injury in adult zebrafish | |
US20230058339A1 (en) | Neurosteroids and enantiomers thereof for the prevention and treatment of neurodegenerative conditions | |
Kim et al. | Carnosine decreases retinal ganglion cell death in a mouse model of optic nerve crushing | |
US20190083455A1 (en) | Methods and compositions for the modulation of beta-endorphin levels | |
KR20150083167A (en) | Pharmaceutical composition comprising methylene blue for treating or preventing sensorineural hearing loss | |
Ruel et al. | Endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) plays a protective effect against noise-induced hearing loss | |
CN106456571B (en) | Composition for treating mechanical neuronal injury | |
Hass | UCP2-Dependent Changes in Mitochondrial Dynamics Protect the Retina from Glaucoma | |
Luo et al. | Dexamethasone protects against arsanilic acid‑induced rat vestibular dysfunction through the BDNF and JNK 1/2 signaling pathways | |
US20220401408A1 (en) | Compositions and methods for rescuing retinal and choroidal structure and function |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20211022 |
|
EEER | Examination request |
Effective date: 20211022 |
|
EEER | Examination request |
Effective date: 20211022 |
|
EEER | Examination request |
Effective date: 20211022 |
|
EEER | Examination request |
Effective date: 20211022 |
|
EEER | Examination request |
Effective date: 20211022 |
|
EEER | Examination request |
Effective date: 20211022 |